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1.  First Outbreak of West Nile Virus Neuroinvasive Disease in Humans, Croatia, 2012 
Between September 6 and 21, 2012, seven human cases of West Nile virus (WNV) neuroinvasive infection were laboratory confirmed in Croatia. The median patient age was 62 years (range 48–77). Five patients presented with meningoencephalitis and two patients with meningoencephalitis followed by acute flaccid paralysis. Four of them had an underlying disease (hypertension). Using enzyme-linked immunosorbent assay (ELISA), WNV-specific immunoglobulin M (IgM) and IgG antibodies of low avidity were detected in six patients, whereas one showed only IgM antibodies. All samples were confirmed using plaque-reduction neutralization and microneutralization tests. Five patients recovered fully. Before human cases were reported, acute asymptomatic WNV infection was demonstrated by detection of IgM antibodies in sentinel horses. Moreover, an increased WNV IgG seropositivity in horses was detected in counties where human cases occurred. Adulticidal and larvicidal treatments were administered immediately in the respective places of residence. The end of the warm season contributed to the fact that there were no new cases of WNV disease recorded.
PMCID: PMC3880908  PMID: 24283515
West Nile virus; Outbreak; Croatia
2.  Infections by Leptospira interrogans, Seoul Virus, and Bartonella spp. Among Norway Rats (Rattus norvegicus) from the Urban Slum Environment in Brazil 
Norway rats (Rattus norvegicus) are reservoir hosts for zoonotic pathogens that cause significant morbidity and mortality in humans. Studies evaluating the prevalence of zoonotic pathogens in tropical Norway rat populations are rare, and data on co-infection with multiple pathogens are nonexistent. Herein, we describe the prevalence of leptospiral carriage, Seoul virus (SEOV), and Bartonella spp. infection independently, in addition to the rates of co-infection among urban, slum-dwelling Norway rats in Salvador, Brazil, trapped during the rainy season from June to August of 2010. These data were complemented with previously unpublished Leptospira and SEOV prevalence information collected in 1998. Immunofluorescence staining of kidney impressions was used to identify Leptospira interrogans in 2010, whereas isolation was used in 1998, and western blotting was used to detect SEOV antibodies in 2010, whereas enzyme-linked immunosorbent assay (ELISA) was used in 1998: in 2010, Bartonella spp. were isolated from a subsample of rats. The most common pathogen in both years was Leptospira spp. (83%, n=142 in 1998, 63%, n=84 in 2010). SEOV was detected in 18% of individuals in both 1998 and 2010 (n=78 in 1998; n=73 in 2010), and two species of Bartonella were isolated from 5 of 26 rats (19%) tested in 2010. The prevalence of all agents increased significantly with rat mass/age. Acquisition of Leptospira spp. occurred at a younger mass/age than SEOV and Bartonella spp. infection, suggesting differences in the transmission dynamics of these pathogens. These data indicate that Norway rats in Salvador serve as reservoir hosts for all three of these zoonotic pathogens and that the high prevalence of leptospiral carriage in Salvador rats poses a high degree of risk to human health.
PMCID: PMC3880909  PMID: 24359425
Leptospira interrogans; Seoul virus; Bartonella spp.; Norway rats (Rattus norvegicus); Brazil
3.  Humans and Cattle: A Review of Bovine Zoonoses 
Infectious disease prevention and control has been among the top public health objectives during the last century. However, controlling disease due to pathogens that move between animals and humans has been challenging. Such zoonotic pathogens have been responsible for the majority of new human disease threats and a number of recent international epidemics. Currently, our surveillance systems often lack the ability to monitor the human–animal interface for emergent pathogens. Identifying and ultimately addressing emergent cross-species infections will require a “One Health” approach in which resources from public veterinary, environmental, and human health function as part of an integrative system. Here we review the epidemiology of bovine zoonoses from a public health perspective.
PMCID: PMC3880910  PMID: 24341911
Cattle zoonoses; Emerging pathogens; Occupational exposure; Epidemiology; Public health
4.  Identification of Bartonellae in the Soft Tick Species Ornithodoros sonrai in Senegal 
Ticks, belonging to the soft ticks species Ornithodorus sonrai, have been collected from six sites in Senegal and were tested for the presence of Bartonella spp. Initial screening by PCR revealed the presence of these bacteria in ticks from two villages, Soulkhou Thissé (5/8, 62.5%) and Maka Gouye (1/24, 4.2%). Three bacterial strains were isolated from live ticks, and the genetic characterization of these strains suggests that they belong to two previously unknown species. The pathogenicity of these two new species of Bartonella is not yet known. The new isolates described here are the first strains of Bartonella spp. from soft ticks and the first isolates from any arthropod species in Africa.
PMCID: PMC3880920  PMID: 24359424
Ornithodorus sonrai; Soft ticks; Bartonella; Senegal
5.  Serological Investigation of Wild Boars (Sus scrofa) and Red Foxes (Vulpes vulpes) As Indicator Animals for Circulation of Francisella tularensis in Germany 
Tularemia outbreaks in humans have recently been reported in many European countries, but data on the occurrence in the animal population are scarce. In North America, seroconversion of omnivores and carnivores was used as indicator for the presence of tularemia, for the European fauna, however, data are barely available. Therefore, the suitability of wild boars (Sus scrofa) and red foxes (Vulpes vulpes) as indicators for the circulation of F. tularensis in Germany was evaluated. Serum samples from 566 wild boars and 457 red foxes were collected between 1995 and 2012 in three federal states in Central Germany (Hesse, Saxony-Anhalt, and Thuringia). The overall rate of seropositive animals was 1.1% in wild boars and 7.4% in red foxes. In conclusion, serological examination of red foxes is recommended, because they can be reliably used as indicator animals for the presence of F. tularensis in the environment.
PMCID: PMC3880921  PMID: 24359418
Francisella tularensis; Fox; Tularemia; Wild boar; Wildlife; Zoonoses
6.  Seroepidemiological Survey of Q Fever and Brucellosis in Kurdistan Province, Western Iran 
Given that the there is little information about the current status of brucellosis and Q fever in most parts of Iran, the aim of this study was to assay the seroprevalence of these two diseases in high-risk populations of Kurdistan Province in western Iran. Two hundred fifty sera samples were collected from hunters and their families, butchers, health care workers, and those referred to medical diagnostic laboratories in the southwestern regions of Kurdistan Province. Sera were tested to detect specific immunoglobulin G (IgG) antibodies against brucellosis and Coxiella burnetii (phase I and II). The seroprevalence of brucellosis and Q fever (C. burnetii IgG phase I and II) was 6.4% and 27.83% (20% and 14.52%), respectively. The highest seroprevalence of Q fever (38%) and brucellosis (12%) was seen in butchers, who handled cattle, sheep, and goats during their work. Age had a significant positive association with Q fever seropositivity (p=0.04). The seroprevalence of Q fever was higher in those people who had been in employment for more than 10 years (21.88%) compared to others (7.79%) (p=0.02). The keeping of animals (p=0.03), hunting and eating the meat of wild animals (p=0.02), and not disinfecting hands and faces after working (for health care workers and butchers) (p=0.02) were risk factors for Q fever seropositivity. This study showed a relatively high seroprevalence of brucellosis and Q fever in high-risk populations of Kurdistan Province. It is suggested that complementary studies be carried out in other parts of western Iran to clarify the epidemiological aspects of these diseases.
PMCID: PMC3880925  PMID: 24359427
Coxiella burnetii; Brucellosis; Seroprevalence–Kurdistan; Risk factor
7.  Bartonella Species and Trombiculid Mites of Rats from the Mekong Delta of Vietnam 
A survey of Bartonella spp. from 275 rats purchased in food markets (n=150) and trapped in different ecosystems (rice field, forest, and animal farms) (n=125) was carried out during October, 2012–March, 2013, in the Mekong Delta of Vietnam. The overall Bartonella spp. prevalence detected by culture and PCR in blood was 14.9% (10.7–19.1%), the highest corresponding to Rattus tanezumi (49.2%), followed by Rattus norvegicus (20.7%). Trapped rats were also investigated for the presence and type of chiggers (larvae of trombiculid mites), and Bartonella spp. were investigated on chigger pools collected from each rat by RT-PCR. A total of five Bartonella spp. were identified in rats, three of which (B. elizabethae, B. rattimassiliensis, and B. tribocorum) are known zoonotic pathogens. Among trapped rats, factors independently associated with increased prevalence of Bartonella spp. included: (1) Rat species (R. tanezumi); (2) the number of Trombiculini–Blankaartia and Schoengastiini–Ascoschoengastia mites found on rats; and (3) the habitat of the rat (i.e., forest/fields vs. animal farms). The prevalence of Bartonella infection among chiggers from Bartonella spp.–positive R. tanezumi rats was 5/25 (25%), compared with 1/27 (3.7%) among Bartonella spp.–negative R. tanezumi rats (relative risk [RR]=5.4, 95% confidence interval [CI] 0.68–43.09). The finding of Bartonella spp.–positive chiggers on Bartonella spp.–negative rats is strongly suggestive of a transovarial transmission cycle. Rats are ubiquitous in areas of human activity and farms in the Mekong Delta; in addition, trapping and trading of rats for food is common. To correctly assess the human risks due to rat trapping, marketing, and carcass dressing, further studies are needed to establish the routes of transmission and cycle of infection. The widespread presence of these zoonotic pathogens in rats and the abundance of human—rat interactions suggest that surveillance efforts should be enhanced to detect any human cases of Bartonella infection that may arise.
PMCID: PMC4307099  PMID: 25629779
Bartonella; Rattus; Chiggers; Vietnam
8.  How Important Are Rats As Vectors of Leptospirosis in the Mekong Delta of Vietnam? 
Leptospirosis is a zoonosis known to be endemic in the Mekong Delta of Vietnam, even though clinical reports are uncommon. We investigated leptospira infection in rats purchased in food markets during the rainy season (October) (n=150), as well as those trapped during the dry season (February–March) (n=125) in the region using RT-PCR for the lipL32 gene, confirmed by 16S rRNA, as well as by the microscopic agglutination test (MAT). Results were compared with the serovar distribution of human cases referred from Ho Chi Minh City hospitals (2004–2012) confirmed by MAT (n=45). The MAT seroprevalence among rats was 18.3%. The highest MAT seroprevalence corresponded, in decreasing order, to: Rattus norvegicus (33.0%), Bandicota indica (26.5%), Rattus tanezumi (24.6%), Rattus exulans (14.3%), and Rattus argentiventer (7.1%). The most prevalent serovars were, in descending order: Javanica (4.6% rats), Lousiana (4.2%), Copenageni (4.2%), Cynopterie (3.7%), Pomona (2.9%), and Icterohaemorrhagiae (2.5%). A total of 16 rats (5.8%) tested positive by RT-PCR. Overall, larger rats tended to have a higher prevalence of detection. There was considerable agreement between MAT and PCR (kappa=0.28 [0.07–0.49]), although significantly more rats were positive by MAT (McNemar 29.9 (p<0.001). MAT prevalence was higher among rats during the rainy season compared with rats in the dry season. There are no current available data on leptospira serovars in humans in the Mekong Delta, although existing studies suggest limited overlapping between human and rat serovars. Further studies should take into account a wider range of potential reservoirs (i.e., dogs, pigs) as well as perform geographically linked co-sampling of humans and animals to establish the main sources of leptospirosis in the region.
PMCID: PMC4307199  PMID: 25629781
Leptospirosis; Mekong Delta, Vietnam; Rats
9.  Identification of diverse Salmonella Serotypes, Virulotypes, and Antimicrobial Resistance Phenotypes in Waterfowl From Chile 
Vector Borne and Zoonotic Diseases  2013;13(12):884-887.
Salmonella enterica is a pathogen with a wide host-range that presents great concern in developed and developing countries. To determine and characterize Salmonella strains found in Chile's waterfowl, we sampled 758 birds along 2000 km of the Chilean coast. In this sample, 46 isolates from 10 serotypes were detected, several with multidrug resistance phenotypes and different combinations of virulence-associated genes (virulotypes). These results suggest that Salmonella infection in waterfowl in Chile could have impacts on public and animal health.
PMCID: PMC3868272  PMID: 24107205
Salmonella enterica; Waterfowl; Chile; Virulotypes
10.  Empiric Antibiotic Treatment of Erythema Migrans-Like Skin Lesions As a Function of Geography: A Clinical and Cost Effectiveness Modeling Study 
Vector Borne and Zoonotic Diseases  2013;13(12):877-883.
The skin lesion of early Lyme disease, erythema migrans (EM), is so characteristic that routine practice is to treat all such patients with antibiotics. Because other skin lesions may resemble EM, it is not known whether presumptive treatment of EM is appropriate in regions where Lyme disease is rare. We constructed a decision model to compare the cost and clinical effectiveness of three strategies for the management of EM: Treat All, Observe, and Serology as a function of the probability that an EM-like lesion is Lyme disease. Treat All was found to be the preferred strategy in regions that are endemic for Lyme disease. Where Lyme disease is rare, Observe is the preferred strategy, as presumptive treatment would be expected to produce excessive harm and increased costs. Where Lyme disease is rare, clinicians and public health officials should consider observing patients with EM-like lesions who lack travel to Lyme disease-endemic areas.
PMCID: PMC3868279  PMID: 24107201
Lyme disease; Lyme borreliosis; Erythema migrans; Southern tick-associated rash illness (STARI); Borrelia burgdorferi; Ixodes scapularis; Deer tick; Black-legged tick; Amblyomma americanum; Lone star tick; Antibiotics; Doxycycline; Amoxicillin; Cost-effectiveness; Decision analysis
11.  Identification of Dengue Fever Cases in Houston, Texas, with Evidence of Autochthonous Transmission Between 2003 and 2005 
Vector Borne and Zoonotic Diseases  2013;13(12):835-845.
Houston, Texas, maintains an environment conducive to dengue virus (DENV) emergence; however, surveillance is passive and diagnostic testing is not readily available. To determine if DENV is present in the area, we tested 3768 clinical specimens (2138 cerebrospinal fluid [CSF] and 1630 serum) collected from patients with suspected mosquito-borne viral disease between 2003 and 2005. We identified 47 immunoglobulin M (IgM)-positive dengue cases, including two cases that were positive for viral RNA in serum for dengue serotype 2. The majority of cases did not report any history of travel outside the Houston area prior to symptom onset. The epidemic curve suggests an outbreak occurred in 2003 with continued low-level transmission in 2004 and 2005. Chart abstractions were completed for 42 of the 47 cases; 57% were diagnosed with meningitis and/or encephalitis, and 43% met the case definition for dengue fever. Two of the 47 cases were fatal, including one with illness compatible with dengue shock syndrome. Our results support local transmission of DENV during the study period. These findings heighten the need for dengue surveillance in the southern United States.
PMCID: PMC3868290  PMID: 24107180
Dengue virus; West Nile virus; Surveillance; Meningitis; IgM enzyme-linked immunosorbent assay; RT-PCR
12.  Molecular Identification of Vertebrate and Hemoparasite DNA Within Mosquito Blood Meals From Eastern North Dakota 
Vector Borne and Zoonotic Diseases  2013;13(11):818-824.
To understand local transmission of vector-borne diseases, it is important to identify potential vectors, characterize their host feeding patterns, and determine if vector-borne pathogens are circulating within the region. This study simultaneously investigated these aspects of disease transmission by collecting engorged mosquitoes within two rural study sites in the central Red River Valley of North Dakota. Mosquitoes were identified, midguts were excised, and the blood was expelled from the midguts. DNA was extracted from blood meals and subjected to PCR and direct sequencing to identify the vertebrate origin of the blood. Using different primer sets, PCR was used to screen for two types of vector-borne pathogens, filarioid nematodes and hemosporidian parasites. White-tailed deer were the primary source of blood meals for the eight aedine mosquito species collected. None of the 288 deer-derived blood meals contained filarioid or hemosporidian DNA. In contrast, 18 of 32 Culex tarsalis and three of three Cx. pipiens blood meals contained avian blood, representing eight different species of birds. Of 24 avian-derived blood meals examined, 12 contained Plasmodium DNA, three of which also contained Leucocytozoon DNA (i.e., dual infection). Potential confounding effects resulting from parasite acquisition and development from previous blood meals (e.g., oocysts) were eliminated because host blood had been removed from the midguts prior to DNA extraction. Thus, specific parasite lineages/species could be unequivocally linked to specific vertebrate species. By combining mosquito identification with molecular techniques for identifying blood meal source and pathogens, a relatively small sample of engorged mosquitoes yielded important new information about mosquito feeding patterns and hemosporidia infections in birds. Thorough analyses of wild-caught engorged mosquitoes and other arthropods represent a powerful tool in understanding the local transmission of vector-borne and zoonotic diseases.
PMCID: PMC3822360  PMID: 24107213
Mosquito; Blood meal; Xenomonitoring; Plasmodium; Leucocytozoon; PCR
13.  Quantitative PCR for Detection of Babesia microti in Ixodes scapularis Ticks and in Human Blood 
Vector Borne and Zoonotic Diseases  2013;13(11):784-790.
Babesia microti, the primary cause of human babesiosis in the United States, is transmitted by Ixodes scapularis ticks; transmission may also occur through blood transfusion and transplacentally. Most infected people experience a viral-like illness that resolves without complication, but those who are immunocompromised may develop a serious and prolonged illness that is sometimes fatal. The geographic expansion and increasing incidence of human babesiosis in the northeastern and midwestern United States highlight the need for high-throughput sensitive and specific assays to detect parasites in both ticks and humans with the goals of improving epidemiological surveillance, diagnosis of acute infections, and screening of the blood supply. Accordingly, we developed a B. microti-specific quantitative PCR (qPCR) assay (named BabMq18) designed to detect B. microti DNA in tick and human blood samples using a primer and probe combination that targets the 18S rRNA gene of B. microti. This qPCR assay was compared with two nonquantitative B. microti PCR assays by testing tick samples and was found to exhibit higher sensitivity for detection of B. microti DNA. The BabMq18 assay has a detection threshold of 10 copies per reaction and does not amplify DNA in I. scapularis ticks infected with Babesia odocoilei, Borrelia burgdorferi, Borrelia miyamotoi, or Anaplasma phagocytophilum. This highly sensitive and specific qPCR assay can be used for detection of B. microti DNA in both tick and human samples. Finally, we report the prevalence of B. microti infection in field-collected I. scapularis nymphs from three locations in southern New England that present disparate incidences of human babesiosis.
PMCID: PMC3822370  PMID: 24107203
Babesia microti; Ixodes scapularis; Babesiosis; Quantitative PCR; Babesia odocoilei; Ticks
14.  Limited Spillover to Humans from West Nile Virus Viremic Birds in Atlanta, Georgia 
Vector Borne and Zoonotic Diseases  2013;13(11):812-817.
West Nile Virus (WNV) is a mosquito-borne pathogen that impacts the health of its passerine bird hosts as well as incidentally infected humans in the United States. Intensive enzootic activity among the hosts and vectors does not always lead to human outbreaks, as is the situation throughout much of the southeastern United States. In Georgia, substantial yearly evidence of WNV in the mosquito vectors and avian hosts since 2001 has only led to 324 human cases. Although virus has been consistently isolated from mosquitoes trapped in Atlanta, GA, little is known about viral activity among the passerine hosts. A possible reason for the suppression of WNV spillover to humans is that viremic birds are absent from high human-use areas of the city. To test this hypothesis, multiseason, multihabitat, longitudinal WNV surveillance for active WNV viremia was conducted within the avian host community of urban Atlanta by collection of blood samples from wild passerine birds in five urban microhabitats. WNV was isolated from the serum of six blood samples collected from 630 (0.95%) wild passerine birds in Atlanta during 2010–2012, a proportion similar to that found in the Chicago, IL, area in 2005, when over 200 human cases were reported. Most of the viremic birds were Northern Cardinals, suggesting they may be of particular importance to the WNV transmission cycle in Georgia. Results indicated active WNV transmission in all microhabitats of urban Atlanta, except in the old-growth forest patches. The number of viremic birds was highest in Zoo Atlanta, where 3.5% of samples were viremic. Although not significant, these observations may suggest a possible transmission reduction effect of urban old-growth forests and a potential role in WNV amplification for Zoo Atlanta. Overall, spillover to humans remains a rare occurrence in urban Atlanta settings despite active WNV transmission in the avian population.
PMCID: PMC3822371  PMID: 24107200
West Nile Virus; Viremia; Spillover; Northern Cardinal; Urban; Zoo; Atlanta, Georgia
15.  Molecular Phylogenetic Analysis of Orientia tsutsugamushi Based on the groES and groEL Genes 
Vector Borne and Zoonotic Diseases  2013;13(11):825-829.
DNA sequences encoding the GroES and GroEL proteins of Orientia tsutsugamushi were amplified by the PCR and sequenced. Pairwise alignment of full-length groES and groEL gene sequences indicated high sequence similarity (90.4–100% and 90.3–100%) in O. tsutsugamushi, suggesting that these genes are good candidates for the molecular diagnosis and phylogenetic analysis of scrub typhus. Comparisons of the 56-kD type-specific antigen (TSA) protein gene and the groES and groEL genes showed that genotypes based on the 56-kD TSA gene were not related to a cluster containing the groES and groEL genes in a dendrogram, suggesting that a gene rearrangement may be associated with homologous recombination in mites.
PMCID: PMC3822374  PMID: 24107204
Orientia tsutsugamushi; groES and groEL genes; Phylogeny; Scrub typhus; Japan
16.  The Role of Game (Wild Boar and Roe Deer) in the Spread of Tick-Borne Encephalitis in the Czech Republic 
Vector Borne and Zoonotic Diseases  2014;14(11):801-807.
In the Czech Republic, the incidence of human tick-borne encephalitis (TBE) has been increasing over the last two decades. At the same time, populations of game have also shown an upward trend. In this country, the ungulate game is the main host group of hosts for Ixodes ricinus female ticks. This study examined the potential contribution of two most widespread game species (roe deer [Capreolus capreolus] and wild boar [Sus scrofa]) to the high incidence of TBE in the Czech Republic, using the annual numbers of culls as a proxy for the game population. This was an ecological study, with annual figures for geographical areas—municipalities with extended competence (MEC)—used as units of analysis. Between 2003 and 2011, a total of 6213 TBE cases were reported, and 1062,308 roe deer and 989,222 wild boars were culled; the culls of roe deer did not demonstrate a clear temporal trend, but wild boar culls almost doubled (from 77,269 to 143,378 per year). Statistical analyses revealed a positive association between TBE incidence rate and the relative number of culled wild boars. In multivariate analyses, a change in the numbers of culled wild boars between the 25th and 75th percentile was associated with TBE incidence rate ratio of 1.23 (95% confidence interval 1.07–1.41, p=0.003). By contrast, the association of TBE with culled roe deer was not statistically significant (p=0.481). The results suggest that the size of the wild boar population may have contributed to the current high levels and the rising trend in incidence of TBE, whereas the regulated population of roe deer does not seem to be implicated in recent geographical or temporal variations in TBE in the Czech Republic.
PMCID: PMC4238255  PMID: 25409271
Tick-borne encephalitis; Incidence; Game; Wild boar; Roe deer
17.  Lobomycosis: Risk of Zoonotic Transmission from Dolphins to Humans 
Vector Borne and Zoonotic Diseases  2013;13(10):689-693.
Lobomycosis, a fungal disease of the skin and subcutaneous tissues caused by Lacazia loboi, is sometimes referred to as a zoonotic disease because it affects only specific delphinidae and humans; however, the evidence that it can be transferred directly to humans from dolphins is weak. Dolphins have also been postulated to be responsible for an apparent geographic expansion of the disease in humans. Morphological and molecular differences between the human and dolphin organisms, differences in geographic distribution of the diseases between dolphins and humans, the existence of only a single documented case of presumed zoonotic transmission, and anecdotal evidence of lack of transmission to humans following accidental inoculation of tissue from infected dolphins do not support the hypothesis that dolphins infected with L. loboi represent a zoonotic hazard for humans. In addition, the lack of human cases in communities adjacent to coastal estuaries with a high prevalence of lobomycosis in dolphins, such as the Indian River Lagoon in Florida (IRL), suggests that direct or indirect transmission of L. loboi from dolphins to humans occurs rarely, if at all. Nonetheless, attention to personal hygiene and general principals of infection control are always appropriate when handling tissues from an animal with a presumptive diagnosis of a mycotic or fungal disease.
PMCID: PMC3787463  PMID: 23919604
Lobomycosis; Lacazia loboi; Zoonoses; Dolphins; Marine mammals; Mycoses
18.  West Nile Virus Equine Serosurvey in the Czech and Slovak Republics 
Vector Borne and Zoonotic Diseases  2013;13(10):733-738.
A serological survey for West Nile virus (WNV) infection involved 395 horses from 43 administrative districts of the Czech Republic (163 animals) and 29 districts of Slovakia (232 animals), sampled between 2008 and 2011. Using a plaque-reduction neutralization microtest, antibodies to WNV were not detected in any horse from the Czech Republic, whereas 19 nonvaccinated horses from Slovakia had specific antibodies to WNV (no cross-reactions were observed with tick-borne encephalitis and Usutu flaviviruses in those animals). The seropositivity rate of nonvaccinated horses in Slovakia was 8.3% (95% confidence interval [CI] 4.7–11.9%), and autochthonous local infection with WNV occurred at least in 11, i.e., 4.8% (95% CI 2.0–7.6%) of the animals. All seropositive horses lived in six lowland districts of southern Slovakia; overall, 15.1% (95% CI 8.8–21.4%) of 126 nonvaccinated horses were seropositive in those districts, situated relatively closely to the border with Hungary, i.e., the country where WNV disease cases have been reported in birds, horses and humans since 2003.
PMCID: PMC3787466  PMID: 23919605
Flavivirus; Mosquito-borne virus; West Nile virus; Neutralizing antibodies; Horses
19.  Assessment of Transcriptional Activity of Borrelia burgdorferi and Host Cytokine Genes During Early and Late Infection in a Mouse Model 
Vector Borne and Zoonotic Diseases  2013;13(10):694-711.
Differential gene expression by Borrelia burgdorferi spirochetes during mammalian infection facilitates their dissemination as well as immune evasion. Modulation of gene transcription in response to host immunity has been documented with the outer surface protein C, but the influence of transcription of other genes is largely unknown. A low-density array (LDA) was developed to study transcriptional activity of 43 B. burgdorferi genes and 19 host genes that may be involved in various host–agent interactions. Gene transcription in heart, joint, and muscle tissue was compared in immunocompetent C3H and immunodeficient C3H-scid mice during early (3 weeks) and late (2 months) B. burgdorferi infection. Among all tissue types, levels of relative transcription of over 80% of B. burgdorferi genes tested were one- to nine-fold less in C3H mice compared to C3H-scid mice. At the later time point, all genes were transcribed in C3H-scid mice, whereas transcription of 16 genes out of 43 tested was not detected in analyzed tissues of C3H mice. Our data suggest that during infection of immunocompetent mice, a majority of B. burgdorferi genes tested are downregulated in response to acquired host immunity. LDA revealed variable patterns of host gene expression in different tissues and at different intervals in infected mice. Higher levels of relative expression for IL-10 during both early and late infection were detected in heart base, and it was unchanged in the tibiotarsal joint. Comparative analysis of B. burgdorferi and host genes transcriptional activity revealed that increased flaB mRNA during early infection was followed by increases of CCL7, CCL8, interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α) in all assessed tissue types. LDA represents a valuable approach for sensitive and quantitative gene transcription profiling and for understanding Lyme borreliosis.
PMCID: PMC3787468  PMID: 23930938
Borrelia burgdorferi; Cytokine; Transcription; Mouse; Low-density array
20.  High Prevalence of Rickettsia africae Variants in Amblyomma variegatum Ticks from Domestic Mammals in Rural Western Kenya: Implications for Human Health 
Vector Borne and Zoonotic Diseases  2014;14(10):693-702.
Tick-borne spotted fever group (SFG) rickettsioses are emerging human diseases caused by obligate intracellular Gram-negative bacteria of the genus Rickettsia. Despite being important causes of systemic febrile illnesses in travelers returning from sub-Saharan Africa, little is known about the reservoir hosts of these pathogens. We conducted surveys for rickettsiae in domestic animals and ticks in a rural setting in western Kenya. Of the 100 serum specimens tested from each species of domestic ruminant 43% of goats, 23% of sheep, and 1% of cattle had immunoglobulin G (IgG) antibodies to the SFG rickettsiae. None of these sera were positive for IgG against typhus group rickettsiae. We detected Rickettsia africae–genotype DNA in 92.6% of adult Amblyomma variegatum ticks collected from domestic ruminants, but found no evidence of the pathogen in blood specimens from cattle, goats, or sheep. Sequencing of a subset of 21 rickettsia-positive ticks revealed R. africae variants in 95.2% (20/21) of ticks tested. Our findings show a high prevalence of R. africae variants in A. variegatum ticks in western Kenya, which may represent a low disease risk for humans. This may provide a possible explanation for the lack of African tick-bite fever cases among febrile patients in Kenya.
PMCID: PMC4208559  PMID: 25325312
Ambylomma variegatum; Rickettsia africae; Tick-borne spotted fever group; African tick-bite fever
21.  Coxiella burnetii in Central Italy: Novel Genotypes Are Circulating in Cattle and Goats 
Vector Borne and Zoonotic Diseases  2014;14(10):710-715.
Genotyping of bacteria is critical for diagnosis, treatment, and epidemiological surveillance. Coxiella burnetii, the etiological agent of Q fever, has been recognized to have a potential for bioterrorism purposes. Because few serosurveys have been conducted in Italy, there is still limited information about the distribution of this pathogen in natural conditions. In this paper, we describe the genotyping of C. burnetii strains by multispacer sequence typing (MST) detected in cattle and goat farms in the Abruzzi region of Italy. Biological samples (milk, aborted fetus) positive for C. burnetii DNA were sequenced in the spacer regions and compared with those already publicly available ( The MST profile of C. burnetii detected in milk samples demonstrated the presence of a new allele, whereas the C. burnetii spacer sequences from fetus and milk goat samples displayed a new allelic combination. The results suggest the circulation of novel genotypes of C. burnetii in Italy.
PMCID: PMC4208599  PMID: 25325314
Coxiella burnetii; Genotyping; Multispacer sequence typing; Cattle–Goat; Italy
22.  Early Detection of Trichinella spiralis in Muscle of Infected Mice by Real-Time Fluorescence Resonance Energy Transfer PCR 
Real-time fluorescence resonance energy transfer (FRET) PCR and melting curve analysis using newly developed fluorophore-labeled hybridization probes were applied for the detection of Trichinella spiralis DNA in muscle of mice following oral inoculation with 300 T. spiralis larvae. The developed assay could detect and differentiate T. spiralis, Trichinella papuae, and Trichinella pseudospiralis DNAs by the different melting temperatures (Tm). The assay had a detection limit of 5×102 positive control plasmid copies, which was equivalent to 1 ng of T. spiralis DNA spiked into 250 mg of muscle sample. No fluorescence signal was detected when the technique was applied to the DNA of 27 parasites other than Trichinella spp. The assay could detect T. spiralis DNA in muscle at 7, 14, and 21 days postinoculation. The range, mean±standard deviation, and median of the Tm values of all positive muscle tissue samples were 60.4–60.8, 60.6±0.2, and 60.5, respectively. This assay provides an effective tool for the specific, sensitive, and high-throughput detection of T. spiralis DNA in muscle during the early stage of infection. In addition, the technique can be useful for epidemiologic surveillance in naturally infected wildlife.
PMCID: PMC3777295  PMID: 23808975
Trichinella spiralis; Real-time FRET PCR; Detection; Muscle
23.  Lymphatic Filariasis Transmission Risk Map of India, Based on a Geo-Environmental Risk Model 
The strategy adopted by a global program to interrupt transmission of lymphatic filariasis (LF) is mass drug administration (MDA) using chemotherapy. India also followed this strategy by introducing MDA in the historically known endemic areas. All other areas, which remained unsurveyed, were presumed to be nonendemic and left without any intervention. Therefore, identification of LF transmission risk areas in the entire country has become essential so that they can be targeted for intervention. A geo-environmental risk model (GERM) developed earlier was used to create a filariasis transmission risk map for India. In this model, a Standardized Filariasis Transmission Risk Index (SFTRI, based on geo-environmental risk variables) was used as a predictor of transmission risk. The relationship between SFTRI and endemicity (historically known) of an area was quantified by logistic regression analysis. The quantified relationship was validated by assessing the filarial antigenemia status of children living in the unsurveyed areas through a ground truth study. A significant positive relationship was observed between SFTRI and the endemicity of an area. Overall, the model prediction of filarial endemic status of districts was found to be correct in 92.8% of the total observations. Thus, among the 190 districts hitherto unsurveyed, as many as 113 districts were predicted to be at risk, and the remaining at no risk. The GERM developed on geographic information system (GIS) platform is useful for LF spatial delimitation on a macrogeographic/regional scale. Furthermore, the risk map developed will be useful for the national LF elimination program by identifying areas at risk for intervention and for undertaking surveillance in no-risk areas.
PMCID: PMC3777552  PMID: 23808973
Lymphatic filariasis; Transmission Risk Map; GERM; GIS; India
24.  Rift Valley Fever Virus Structural and Nonstructural Proteins: Recombinant Protein Expression and Immunoreactivity Against Antisera from Sheep 
The Rift Valley fever virus (RVFV) encodes the structural proteins nucleoprotein (N), aminoterminal glycoprotein (Gn), carboxyterminal glycoprotein (Gc), and L protein, 78-kD, and the nonstructural proteins NSm and NSs. Using the baculovirus system, we expressed the full-length coding sequence of N, NSs, NSm, Gc, and the ectodomain of the coding sequence of the Gn glycoprotein derived from the virulent strain of RVFV ZH548. Western blot analysis using anti-His antibodies and monoclonal antibodies against Gn and N confirmed expression of the recombinant proteins, and in vitro biochemical analysis showed that the two glycoproteins, Gn and Gc, were expressed in glycosylated form. Immunoreactivity profiles of the recombinant proteins in western blot and in indirect enzyme-linked immunosorbent assay against a panel of antisera obtained from vaccinated or wild type (RVFV)-challenged sheep confirmed the results obtained with anti-His antibodies and demonstrated the suitability of the baculo-expressed antigens for diagnostic assays. In addition, these recombinant proteins could be valuable for the development of diagnostic methods that differentiate infected from vaccinated animals (DIVA).
PMCID: PMC3777644  PMID: 23962238
Rift Valley fever virus; Structural proteins; Nonstructural proteins; Immunoreactivity profiles; Sheep
25.  Molecular detection of Rickettsia felis and Candidatus Rickettsia Asemboensis in Fleas from Human Habitats, Asembo, Kenya 
The flea-borne rickettsioses murine typhus (Rickettsia typhi) and flea-borne spotted fever (FBSF) (Rickettsia felis) are febrile diseases distributed among humans worldwide. Murine typhus has been known to be endemic to Kenya since the 1950s, but FBSF was only recently documented in northeastern (2010) and western (2012) Kenya. To characterize the potential exposure of humans in Kenya to flea-borne rickettsioses, a total of 330 fleas (134 pools) including 5 species (Xenopsylla cheopis, Ctenocephalides felis, Ctenocephalides canis, Pulex irritans, and Echidnophaga gallinacea) were collected from domestic and peridomestic animals and from human dwellings within Asembo, western Kenya. DNA was extracted from the 134 pooled flea samples and 89 (66.4%) pools tested positively for rickettsial DNA by 2 genus-specific quantitative real-time PCR (qPCR) assays based upon the citrate synthase (gltA) and 17-kD antigen genes and the Rfelis qPCR assay. Sequences from the 17-kD antigen gene, the outer membrane protein (omp)B, and 2 R. felis plasmid genes (pRF and pRFd) of 12 selected rickettsia-positive samples revealed a unique Rickettsia sp. (n=11) and R. felis (n=1). Depiction of the new rickettsia by multilocus sequence typing (MLST) targeting the 16S rRNA (rrs), 17-kD antigen gene, gltA, ompA, ompB, and surface cell antigen 4 (sca4), shows that it is most closely related to R. felis but genetically dissimilar enough to be considered a separate species provisionally named Candidatus Rickettsia asemboensis. Subsequently, 81 of the 134 (60.4%) flea pools tested positively for Candidatus Rickettsia asemboensis by a newly developed agent-specific qPCR assay, Rasemb. R. felis was identified in 9 of the 134 (6.7%) flea pools, and R. typhi the causative agent of murine typhus was not detected in any of 78 rickettsia-positive pools assessed using a species-specific qPCR assay, Rtyph. Two pools were found to contain both R. felis and Candidatus Rickettsia asemboensis DNA and 1 pool contained an agent, which is potentially new.
PMCID: PMC3741420  PMID: 23675818
Rickettsia; Fleas; PCR; Multilocus sequence typing; Surveillance

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